Tandem mass spectrometry summarizes a broad range of techniques whereby mass selected ions are subjected to a second (or more) level or mass spectrometric analysis. Such is the social and technological importance of tandem mass spectrometry that to date more than a million blood and plasma samples from newborns have been tested for various disorders using such devices. Tandem mass spectrometry is also a central technology for proteomics and other important areas of macromolecular identification, such as drug and metabolite monitoring for forensics.
At present, there are essentially three physical processes by which the internal energy of gas phase ions is raised above the dissociation threshold: 1) collisions with atoms, molecules, or surfaces 2) photodissociation, and 3) dissociative recombination of positively and negatively charged species. Of these methods, collision activated dissociation (CAD), also called collision induced dissociation (CID), is the most widely practiced method. Although collisional activation has many advantages over alternative activation methods, the major limitation of collisional activation is in its ineffectiveness at dissociating high mass ions (such as biomolecules) and molecules with high barriers for dissociation.
The ineffectiveness of CAD for high mass ions stems from a number of factors including 1) inefficient conversion of kinetic to internal energy and 2) increased number of degrees of freedom. In addition, CAD of biological ions often results in the loss of one or more small neutral losses such as water or ammonia with the consequence of uninformative fragmentation patterns. Wide-band excitation has been described recently to attempt to overcome these difficulties, but other problems remain. Significant ion losses, and subsequent decreases in sensitivity are notable in CAD devices because reagent ions and fragment ions are often scattered during, or inefficiently collected, after the collisional processes. Also, as the size of the reagent ion increases, so does the number of fragment ions over which the residual charges must be spread. Fragmentation into a large number of channels leads to decreased sensitivity and may prevent the ability to perform MSn (n>2) fragmentation analyses.
Surface-induced dissociation (SID) has been applied to ion beam, quadrupole, and ICR-type instruments and shows many improvements over CAD for dissociating large biomolecules. However, significant complications arise from surface sputtering, surface reactions, ion losses and collision angle effects.
In the “top down” approach to proteomics, the dissociation of biomolecules in the kDa-MDa mass range is necessary, and this can only be achieved using CAD if it is used in combination with significant proton attachments to effect coulombic repulsions within the biomolecules. A more promising approach to fragmenting large biomolecules has been through electron capture dissociation (ECD) in FT-ICR instruments. This particular technique seems restricted to ICR instruments, however, and may not be applicable to more commonly available mass spectrometers such as quadrupole based systems. There is also the inherent requirement for multiple charges on the reagent ion, as neutralization by an electron reduces the overall charge of the reagent with each capture. For large mono-positive ions, such as dendromers or polymers, ECD may not be applicable.
Moreover, commercial instruments available today typically cannot directly determine the amino acid sequence of large peptides and whole proteins (e.g. >3 kDa). This is primarily due to the difficulty of breaking apart large ions within mass spectrometers, but also due to the inability to control where fragmentation takes place within the bio-ions. If these limitations regarding the fragmentation of large biomolecules could be overcome, biomedical research that depended on protein identification could be considerably accelerated.
Accordingly, there exists a need for additional or complementary methods for dissociating macromolecular ions in the gas phase. This need is especially essential for large biomolecules of interest to human health, national security and forensic applications wherein existing techniques are ineffective for providing conclusive and reproducible results.